1. Field of the Invention
The present invention relates to the photoactivation of proteins by ultraviolet radiation, so that they may be reacted with other chemical entities, including radio-isotopes, to form useful conjugates.
2. Description of the Background Art
A. Radiolabelling of Antibodies
The conventional method of labeling antibodies with technetium (and similar radiometals) is to reduce the disulfide bonds of the antibody with a chemical reducing agent and then react the reduced antibodies with reduced pertechnetate.
Chemical reducing agents are difficult to handle due to their susceptibility to oxidation. They may cause unwanted side reactions on proteins (for example, by reducing carbonyl groups), and it may be difficult to control the extent of the reduction reaction given the need to use strong reducing reagents.
Prior to radiolabelling, the reduced antibodies must be separated from the remaining reducing reagent to stop the reaction, to remove potentially toxic substances (e.g., DTT, if used for reduction), and to avoid complexing technetium and thereby preventing it from labeling the protein. This purification process is time-consuming and may lead to loss of protein, or to re-oxidation of the protein.
Since a source of reduced technetium is required for radiolabeling, a certain amount of reducing agent must be added back to either the reduced protein or to the technetium source itself. The latter implies that the labeling process is necessarily at least two steps.
Rhodes, et al., J. Nucl. Med., 27:685 (1986 and Crockford and Rhodes, U.S. Pat. No. 4,424,200 (1984) teach a method of labelling F(ab').sub.2 fragments of antibodies with the radioisotope Tc-99m. The fragments were incubated overnight with stannous ions (e.g., SnCl.sub.2), a reducing agent, in a phthalate-tartrate buffer. This "pretinning" process converts the dimeric F(abl).sub.2 to monomeric fragments through reduction of the disulfide bonds connecting the heavy chains. The fragments are then reacted with Tc-99m.
Unfortunately, the Rhodes method has several disadvantages. First, it requires a high concentration of stannous ion to reduce the antibody. Nonetheless, Rhodes desires that about two-thirds of the stannous ions be oxidized in the reaction with the protein, the remainder serving to reduce the pertechnetate when it is added later. However, it is difficult to control the extent to which the stannous ions are involved in the reduction of the antibody. If too little stannous ion was provided, the pertechnetate will not be completely reduced by the residuum. If too much stannous ion was furnished, some will complex the Dertechnetate so that it will not bond to the antibody. Consequently, it is customary, after reducing the antibody, to purify out the residual stannous ion, and then add back a small, controlled amount to reduce the pertechnetate. The purification step of course add to the time and expense of the process. See, e.g. the discussion in Bremer, et al, EP Appl. 271,806, "Process for Producing an Organ-Specific Substance Labeled with Technetium-99m".
Rhodes, U.S. Pat. No. 5,078,985 acknowledge that his original pretinning method had "problems associated with lack of purity of the protein, continued reduction of disulfide bonds in the protein, and the formation of additional reduced protein species prior to admixture with sodium tertechnelate. Fewer stannous ions are needed to reduce pertechnetate than to reduce disulfide bonds in proteins; with some proteins, excess stannous ions cause reduction of disulfide bonds and fragmentation of the protein to less than the desired size." Consequently, Rhodes '985, taught that after the initial reduction, whether with stannous ion or a different agent such as dithiothreitol (DTT), the reduced protein should be purified to substantially remove the first reducing agent and impurities, and stannous ion added in a sufficient amount to reduce the radionuclide (the latter to be added in subsequent step).
Because the stannous ion concentration is high, the likelihood of undesired fragmentation of the antibody is increased. This is because a strong reducing agent, such as stannous ion, will reduce disulfide bonds, and the extent of such reduction, and therefore of fragmentation, is a function of concentration. The Crockford and Rhodes process is not readily adjusted to reduce unwanted fragmentation.
While the '200 patent asserts that a pH of 4.5 to 8.5 is "preferred", in our experience the upper limit on pH is actually 6. With a higher pH, stannous ion is precipitated out of solution, and therefore is unavailable for reduction. This is unfortunate, as some proteins, including certain antibodies, are more stable at a higher pH.
Rhodes' method requires long incubation times; the '200 patent calls for reaction for at least 15 hours, more preferably 21 hours. Such long reactions are inherently more vulnerable to mishaps, such as loss of temperature control or nitrogen supply, contamination, etc. Finally, its yields are lackluster; the yield reported in the Crockford '200 patent was 73% radiolabeled IgG. A more typical yield is a litte over 50% as seen in Table 2A. About 17% of Crockford's Tc was in colloidal form. Moreover, about 28% of the Tc on the antibody was "exchangeable".
Shochat, U.S. Pat. No. 5,061,641 uses the same reducing agent and buffer, but suggests that it may be helpful to contact the resulting technetium-labeled antibody with an "exogenous capping ligand", such as a mercaptoalkane or phosphane, to fill the remaining coordination sites of the radiometal and thereby stabilize it. But this patent does not address any of the other disadvantages of the Rhodes method.
B. Photoafffinity Labeling of Proteins
Photoactivatable agents, including arylazides, have been used to immobilize an antigen or antibody on a support. See Kramer, U.S. Pat. No. 4,689,310; Scheebers, U.S. Pat. No. 4,716,122; AU-A-47690/85 (organogen). Dattagupta, U.S. Pat. No. 4,713,326 photochemically coupled a nucleic acid to a substrate. Other molecules have also been insolubilized by this technique. Lingwood, U.S. Pat. No. 4,597,999. Pandey, et al., J. Immunol. Meth., 94:237-47 (1986) conjugated a haptenic primary aromatic amine (3-azido-N-ethylcarbazole) to various proteins to create a synthetic antigen.
Furthermore, one may prepare a "prodrug" or "protoxin" which is converted to the active drug or toxin by photochemical cleavage of a bridging group. Zweig, U.S. Pat. No. 4,202,323; Senter, U.S. Pat. No. 4,625,014; Edelson, U.S. Pat. No. 4,612,007; Reinherz, U.S. Pat. No. 4,443,427 (col. 4).
Photoaffinity labeling of enzymes is also known. Chowdry, Ann. Rev. Biochem., 48:293-305 (1979). In this technique, a probe compound is allowed to interact with a biomatrix in which a specific receptor or binding site is envisaged. Upon formation of the specific complex by their affinity for one another the pair are chemically linked via a reactive group on the probe. This greatly facilitates the identification and characterization of the receptor itself. The great advantage of probes incorporating a photoactive group is that the linkage process may be activated after all non-specific interactions of the probe have been eliminated and thus little or no indiscriminate binding or loss of probe through hydrolysis takes place. These photoaffinity reagents may be prepared either via synthetic routes for simple molecules or via conjugation of a photoactive ligand for more complex macromolecules.
Thus, photoaffinity labeling of peptide hormone binding sites was reported by Galardy, et al., J. Biol. Chem., 249: 350 (1974). The probes employed were aryl azides, which, when photoactivated to yield aryl nitrenes, can label any binding site containing carbon-hydrogen bonds by insertion into the C--H bond.
Biomira (Noujaim), EP Appl. 354,543 teaches the photochemical attachment of chelating groups to biomolecules, such as proteins (especially antibodies). This may be used to indirectly radiolabel a biomolecule with a chelatable ion. A chelating group is first coupled to a photoactivatable functionality. If reacted with an antibody in the presence of light, the "photochelate" will label the antibody. The disclosed activated species are carbenes, nitrenes and free radicals, though only nitrenes are exemplified.
C. Studies of Effect of Ultraviolet Radiation on Proteins
Low intensity ultraviolet irradiation of human gamma globulin has been shown to result in the generation of free sulfhydryl groups and in the aggregation of the protein. Wickens, et al., "Free radical-mediated aggregation of human gamma globulin, Agents and Actions, 11:650 (1981). Disulfide bonds are reduced at random and the resulting free sulfhydryls can re-combine with free thiol groups on other molecules to generate aggregates. Later experiments showed that other sources of oxygen free-radicals, such as a mixture of copper salts and hydrogen peroxide, had similar effects. Wickens, et al., Biochim. Biophys. Acta, 742: 607 (1983). The intrachain, rather than the interchain, disulfide bonds were deemed to be the primary targets since further fragmentation was not seen. According to Lunec, et al., J. Clin. Invest. 76: 2084 (1985), the aromatic amino acids, especially tryptophan and tyrosine, are also attacked. These researchers teach that free radical damage of IgG, through the mechanism of aggregate formation, resulted in the production of rheumatoid factors, i.e., in an autoimmune response to the aggregated antibodies. Thus, it would normally be considered undesirable to expose antibodies, for long enough periods to reduce disulfide bonds, to ultraviolet radiation.
The aforementioned findings with respect to antibodies are consistent with studies of the inactivation of disulfide enzymes (e.g., trypsin) by ultraviolet radiation; cystine disruption is likely to be a major contributing factor. Moreover, the presence of tryptophan or tyrosine residues in a given protein may contribute to the destruction of the cystine residues. K. Dose, "Theoretical Aspects of the U.V. Inactivation of Proteins containing Disulfide Bonds", Photochem. Photobiol. 6, 437-443 (1967); K. Dose, "The Photolysis of Free Cystine in the Presence of Aromatic Amino Acids", Photochem. Photobiol. 8, 331-335 (1968). As a result, ultraviolet irradiation of antibodies, which contain disulfides, is contraindicated, as it may result in loss of activity if disulfide bonds essential to antigen-binding and/or effector function are ruptured.
No admission is made that any reference or statement herein constitutes prior art, and Applicants reserve the right to challenge the accuracy of the nominal publication date of any cited publication, or of the contents thereof.